Breaking up and making up: The secret life of the vacuolar H+ -ATPase

Abstract

The vacuolar ATPase (V-ATPase; V₁ V_o -ATPase) is a large multisubunit proton pump found in the endomembrane system of all eukaryotic cells where it acidifies the lumen of subcellular organelles including lysosomes, endosomes, the Golgi apparatus, and clathrin-coated vesicles. V-ATPase function is essential for pH and ion homeostasis, protein trafficking, endocytosis, mechanistic target of rapamycin (mTOR), and Notch signaling, as well as hormone secretion and neurotransmitter release. V-ATPase can also be found in the plasma membrane of polarized animal cells where its proton pumping function is involved in bone remodeling, urine acidification, and sperm maturation. Aberrant (hypo or hyper) activity has been associated with numerous human diseases and the V-ATPase has therefore been recognized as a potential drug target. Recent progress with moderate to high-resolution structure determination by cryo electron microscopy and X-ray crystallography together with sophisticated single-molecule and biochemical experiments have provided a detailed picture of the structure and unique mode of regulation of the V-ATPase. This review summarizes the recent advances, focusing on the structural and biophysical aspects of the field.

Keywords: V-ATPase; V1Vo-ATPase; X-ray crystallography; cryo electron microscopy; protein structure; protein-protein interactions; reversible disassembly; rotary catalysis; rotary motor enzyme; vacuolar ATPase.

Figure 1

V‐ATPase subunit arrangement and regulation by reversible dissociation. (A) Subunit architecture of yeast V‐ATPase. (B) Regulation by reversible dissociation. In yeast, under conditions of starvation, V₁‐ATPase disengages from the membrane bound V_o and the activity of both sectors is silenced. Activity silencing is accompanied by a large conformational change in V₁'s H subunit (H_CT) and the N‐terminal domain of the V_o a subunit (a _NT). Illustration adapted from Ref. 73.

Figure 2

Overall subunit architecture of holo V‐ATPase. (A) Crystal structure of EhA₃B₃DF (3vr6; a side‐view of the structure is also shown on the right).47 The arrows indicate closed catalytic sites with AMPPNP bound and the arrowhead points to the open site. (B) Crystal structure of ScDF (4rnd).48 (C,D) Crystal structures of ScEGC_head in two conformations (4dl0, 4efa; C_head not shown).49 (E) Crystal structure of ScH (1ho8).50 (F) Crystal structure of ScC (1u7l).51 (G) Crystal structure of the a _NT homolog from M. ruber (Mra; 3rrk).52 (H) Crystal structure of the d homolog from T. thermophilus (TtC; 1r5z).103 (I) Crystal structure of the proteolipid ring from E. hirae (EhK10; 2bl2)53. (J) CryoEM map of ScV₁V_o (emd‐6284)45 with fitted coordinate models of individual subunits and subcomplexes (3j9t).45 The overall model was generated from individual crystal structures of yeast subunits and homology models generated by threading yeast primary sequences into crystal structures of bacterial A‐ATPase subunits.

Figure 3

V‐ATPase mechanism. (A) V‐ATPase consists of two motors, a V₁‐ATPase and a V_o proton channel. (B) ATP hydrolysis at three of the six AB interfaces drives counterclockwise rotation of the central DF rotor (A₃B₃ seen towards the cytosol). (C) Rotation of the central DF rotor drives rotation of the proteolipid ring past the essential arginine residue in a _CT. Protons enter a cytoplasmic half‐channel and, after being carried 360º on lipid exposed glutamate residues on the c subunits, are released into the lumenal half‐channel.

Figure 4

V‐ATPase conformational changes accompanying reversible disassembly. (A) Conformational changes in the auto‐inhibited ScV₁‐ATPase (5d80).71 Upon holo enzyme dissociation, the C‐terminus of subunit H (H_CT) is released from its binding site on a _NT (in V₁V_o) and rotates 150º to bind at the bottom of the A₃B₃ hexamer (in V₁). H_CT binds to the B subunit of the open catalytic site, thereby stabilizing inhibitory ADP in another site.71 The inhibitory loop is in spacefill and the α helix that binds a _NT in V₁V_o is in orange. (B) CryoEM model of holo ScV₁V_o 45 (3j9u). (C) Conformational changes in the V_o proton channel (5tj5)26. Upon release of V₁‐ATPase from the membrane, the N‐terminus of subunit a (a _NT) moves from its ternary interface with C_foot and EG2 to form a new binding interface with subunit d 26, 73, 88 and the c‐ring is positioned so that Glu108 of isoform c″ is in contact with the essential Arg735 in a _CT.

Figure 5

Interactions at the V₁–V_o interface. (A) The affinities of the individual interactions have been measured using isothermal titration calorimetry of recombinant yeast V‐ATPase subunits.48, 96, 97 (B) Spring‐loading of EG3. It has been suggested that the tension in EG3 would prevent re‐forming of the EG2–a _NT(distal)–C_foot ternary interface after one of these interactions is broken to initiate disassembly.49

Figure 6

Model of the structural changes along the steps of holo V‐ATPase disassembly. (A) It has been proposed that disassembly is initiated in state 3 V₁V_o. 26 (B) The ternary a _NT–EG2–Cfoot interaction is destabilized, allowing a _NT to bind subunit d. (C,D) Two uncoupled catalytic steps convert V₁ into the state 2 conformation. (E) Subunit C dissociates and H_CT rotates 150º to bind at the open catalytic site. V₁ is now in state 2 and autoinhibited and V_o is in state 3, also autoinhibited. (F) V₁V_o dissociates. (G) Views towards to bottom of autoinhibited V₁, illustrating H_CT's binding site on A₃B₃DF, and towards the top of V_o, showing the interaction of a _NT with subunit d. (H) Reassembly must involve release of H_CT and a _NT from their autoinhibitory positions and one catalytic turnover of V₁ to return both sectors into matching state 3 conformations. Note that the starting conformation of V₁V_o and the order of the steps are not firmly established. See also Supporting Information Fig. S1 for an animation of the process.